Victor W. Ho

SHIP Represses the Generation of IL-3-Induced M2 Macrophages by Inhibiting IL-4 Production from Basophils

There is a great deal of interest in determining what regulates the generation of classically activated (M1) vs alternatively activated (M2) macrophages (Mφs) because of the opposing effects that these two Mφ subsets have on tumor progression. We show herein that IL-3 and, to a lesser extent, GM-CSF skew murine Mφ progenitors toward an M2 phenotype, especially in the absence of SHIP. Specifically, the addition of these cytokines, with or without M-CSF, to adherence- or lineage-depleted (Lin−) SHIP−/− bone marrow (BM) cells induces high levels of the M2 markers, arginase I, and Ym1 in the resulting mature Mφs. These in vitro-derived mature Mφs also display other M2 characteristics, including an inability to enhance anti-CD3-stimulated splenic T cell secretion of IFN-γ and low IL-12 and high IL-10 production in response to LPS. Not surprisingly, given that IL-3 and GM-CSF utilize STAT5 to trigger many downstream signaling pathways, this M2 phenotype is suppressed when STAT5−/− BM cells are used. Unexpectedly, however, this M2 phenotype is also suppressed when STAT6−/− BM cells are used, suggesting that IL-4- or IL-13-induced signaling might be involved. Consistent with this, we found that IL-3 and GM-CSF stimulate the production of IL-4, especially from SHIP−/− Lin− BM cells, and that neutralizing anti-IL-4 Abs block IL-3-induced M2 skewing. Moreover, we found that basophil progenitors within the Lin− BM are responsible for this IL-3- and GM-CSF-induced IL-4 production, and that SHIP represses M2 skewing not by preventing skewing within Mφs themselves but by inhibiting IL-4 production from basophils.

A low carbohydrate, high protein diet slows tumor growth and prevents cancer initiation.

Since cancer cells depend on glucose more than normal cells, we compared the effects of low carbohydrate (CHO) diets to a Western diet on the growth rate of tumors in mice. To avoid caloric restriction-induced effects, we designed the low CHO diets isocaloric with the Western diet by increasing protein rather than fat levels because of the reported tumor-promoting effects of high fat and the immune-stimulating effects of high protein. We found that both murine and human carcinomas grew slower in mice on diets containing low amylose CHO and high protein compared with a Western diet characterized by relatively high CHO and low protein. There was no weight difference between the tumor-bearing mice on the low CHO or Western diets. Additionally, the low CHO-fed mice exhibited lower blood glucose, insulin, and lactate levels. Additive antitumor effects with the low CHO diets were observed with the mTOR inhibitor CCI-779 and especially with the COX-2 inhibitor Celebrex, a potent anti-inflammatory drug. Strikingly, in a genetically engineered mouse model of HER-2/neu-induced mammary cancer, tumor penetrance in mice on a Western diet was nearly 50% by the age of 1 year whereas no tumors were detected in mice on the low CHO diet. This difference was associated with weight gains in mice on the Western diet not observed in mice on the low CHO diet. Moreover, whereas only 1 mouse on the Western diet achieved a normal life span, due to cancer-associated deaths, more than 50% of the mice on the low CHO diet reached or exceeded the normal life span. Taken together, our findings offer a compelling preclinical illustration of the ability of a low CHO diet in not only restricting weight gain but also cancer development and progression.

SHIP Represses Th2 Skewing by Inhibiting IL-4 Production from Basophils

We report that SHIP−/− mice, compared to SHIP+/+ mice, are Th2 skewed with elevated serum IgE and twice as many splenic CD4+ Th2 cells that, when stimulated with anti-CD3, produce more IL-4 and less IFN-γ. Exploring the reason for this Th2 skewing, we found that freshly isolated SHIP−/− splenic and bone marrow basophils are present in elevated numbers and secrete far more IL-4 in response to IL-3 or to FcεRI stimulation than do WT basophils. These SHIP−/− basophils markedly skew wild-type macrophage colony stimulating factor–derived macrophages toward an M2 phenotype, stimulate OT-II CD4+ Th cells to differentiate into Th2 cells, and trigger SHIP+/+ B cells to become IgE-producing cells. All these effects are completely abrogated with neutralizing anti–IL-4 Ab. Exploring the cell signaling pathways responsible for hyperproduction of IL-4 by SHIP−/− basophils, we found that IL-3–induced activation of the PI3K pathway is significantly enhanced and that PI3K inhibitors, especially a p110α inhibitor, dramatically suppresses IL-4 production from these cells. In vivo studies, in which basophils were depleted from mast cell-deficient SHIP+/+ and SHIP−/− mice, confirmed the central role that basophils play in the Th2 skewing of naive SHIP-deficient mice. Taken together, these studies demonstrate that SHIP is a potent negative regulator of IL-4 production from basophils and thus may be a novel therapeutic target for Th1- and Th2-related diseases.

Macrophages Are More Potent Immune Suppressors Ex Vivo Than Immature Myeloid-Derived Suppressor Cells Induced by Metastatic Murine Mammary Carcinomas

Myeloid-derived suppressor cells (MDSCs) are emerging as potential promoters of metastatic tumor growth, and there is interest in targeting immature MDSCs by inducing their differentiation into more mature myeloid cells. We used all-trans retinoic acid (ATRA) to differentiate MDSCs in mice bearing metastatic 4T1 or 4TO7 murine mammary tumors, and assessed the immune-suppressive mechanisms and potencies of different myeloid cell subpopulations. Metastatic mammary tumors induced the accumulation of distinct populations of immature CD11b+Gr1+F4/80−Ly6CmidLy6G+ MDSCs (“Gr1+ cells”) and mature CD11b+Gr1−F4/80+ cells (“F4/80+ cells”) in metastatic target organs. ATRA triggered the differentiation of Gr1+ cells into F4/80+ cells in the lungs and, unexpectedly, enhanced pulmonary metastatic tumor growth. We found that F4/80+Ly6C−Ly6G− mature macrophages (Mϕs) were up to 30-fold more potent immune suppressors than Gr1+ cells on a per-cell basis, which we postulate may contribute to the increased metastatic growth observed with ATRA treatment. F4/80+ cells and Gr1+ cells used different reactive oxygen species (ROS)–mediated mechanisms of immunosuppression ex vivo, with F4/80+ cells producing higher levels of ROS, which is consistent with their superior immunosuppressive abilities. These data highlight the potent immunosuppressive functions of Mϕs, reveal that Mϕs can suppress T cell responses via ROS production, and suggest that ROS inhibitors may be useful in promoting antitumor immune responses. Our findings also caution against using ATRA to modulate myeloid cell differentiation and function to treat breast cancer metastases in the lung, and support the development of therapeutic strategies to enhance antitumor immunity by targeting myeloid cells as a collective group.

SHIP-Deficient Dendritic Cells, Unlike Wild Type Dendritic Cells, Suppress T Cell Proliferation via a Nitric Oxide-Independent Mechanism

Background Dendritic cells (DCs) not only play a crucial role in activating immune cells but also suppressing them. We recently investigated SHIPs role in murine DCs in terms of immune cell activation and found that TLR agonist-stimulated SHIP−/− GM-CSF-derived DCs (GM-DCs) were far less capable than wild type (WT, SHIP+/+) GM-DCs at activating T cell proliferation. This was most likely because SHIP−/− GM-DCs could not up-regulate MHCII and/or co-stimulatory receptors following TLR stimulation. However, the role of SHIP in DC-induced T cell suppression was not investigated. Methodology/Principal Findings In this study we examined SHIPs role in DC-induced T cell suppression by co-culturing WT and SHIP−/− murine DCs, derived under different conditions or isolated from spleens, with αCD3+ αCD28 activated WT T cells and determined the relative suppressive abilities of the different DC subsets. We found that, in contrast to SHIP+/+ and −/− splenic or Flt3L-derived DCs, which do not suppress T cell proliferation in vitro, both SHIP+/+ and −/− GM-DCs were capable of potently suppressing T cell proliferation. However, WT GM-DC suppression appeared to be mediated, at least in part, by nitric oxide (NO) production while SHIP−/− GM-DCs expressed high levels of arginase 1 and did not produce NO. Following exhaustive studies to ascertain the mechanism of SHIP−/− DC-mediated suppression, we could conclude that cell-cell contact was required and the mechanism may be related to their relative immaturity, compared to SHIP+/+ GM-DCs. Conclusions These findings suggest that although both SHIP+/+ and −/− GM-DCs suppress T cell proliferation, the mechanism(s) employed are different. WT GM-DCs suppress, at least in part, via IFNγ-induced NO production while SHIP−/− GM-DCs do not produce NO and suppression can only be alleviated when contact is prevented.

A low carbohydrate, high protein diet combined with celecoxib markedly reduces metastasis

We recently demonstrated that both murine and human carcinomas grow significantly slower in mice on low carbohydrate (CHO), high protein diets than on isocaloric Western diets and that a further reduction in tumor growth rates occur when the low CHO diets are combined with the cyclooxygenase-2 inhibitor, celecoxib. Following upon these studies, we asked herein what effect low CHO, high protein diets, with or without celecoxib, might have on tumor metastasis. In the highly metastatic 4T1 mouse mammary tumor model, a 15% CHO, high protein diet supplemented with celecoxib (1 g/kg chow) markedly reduced lung metastases. Moreover, in longer-term studies using male Transgenic Adenocarcinoma of the Mouse Prostate mice, which are predisposed to metastatic prostate cancer, the 15% CHO diet, with and without celecoxib (0.3 g/kg chow), gave the lowest incidence of metastases, but a more moderate 25% CHO diet containing celecoxib led to the best survival. Metabolic studies with 4T1 tumors suggested that the low CHO, high protein diets may be forcing tumors to become dependent on amino acid catabolism for survival/growth. Taken together, our results suggest that a combination of a low CHO, high protein diet with celecoxib substantially reduces metastasis.

SHIP represses lung inflammation and inhibits mammary tumor metastasis in BALB/c mice

SH2-containing-inositol-5′-phosphatase (SHIP) is a negative regulator of the phosphatidylinositol-3-kinase pathway in hematopoietic cells and limits the development of leukemias and lymphomas. The potential role of SHIP in solid tumor development and metastasis remains unknown. While SHIP restricts the aberrant development of myeloid cells in C57BL/6 mice, there are conflicting reports regarding the effect of SHIP deletion in BALB/c mice with important consequences for determining the influence of SHIP in different model tumor systems. We generated SHIP−/− BALB/c mice and challenged them with syngeneic non-metastatic 67NR or metastatic 4T1 mammary tumors. We demonstrate that SHIP restricts the development, alternative-activation, and immunosuppressive function of myeloid cells in tumor-free and tumor-bearing BALB/c mice. Tumor-free SHIP−/− BALB/c mice exhibited pulmonary inflammation, myeloid hyperplasia, and M2-polarized macrophages and this phenotype was greatly exacerbated by 4T1, but not 67NR, tumors. 4T1-bearing SHIP−/− mice rapidly lost weight and died from necrohemorrhagic inflammatory pulmonary disease, characterized by massive infiltration of pulmonary macrophages and myeloid-derived suppressor cells that were more M2-polarized and immunosuppressive than wild-type cells. Importantly, while SHIP loss did not affect primary tumor growth, 4T1-bearing SHIP−/− mice had 7.5-fold more metastatic tumor cells in their lungs than wild-type mice, consistent with the influence of immunosuppressive myeloid cells on metastatic growth. Our findings identify the hematopoietic cell-restricted protein SHIP as an intriguing target to influence the development of solid tumor metastases, and support development of SHIP agonists to prevent the accumulation of immunosuppressive myeloid cells and tumor metastases in the lungs to improve treatment of metastatic breast cancer.

The Src Homology 2 Containing Inositol 5′ Phosphatases

Publisher Summary This chapter focuses on the relative structure, expression, and biological roles of Src homology 2 containing inositol 5′-phosphatase (SHIP) 1 and SHIP2 in various cell types, and the therapeutic potential of targeting these two phospholipid phosphatases. The phosphatidylinositol (PI)-3-kinase (PI3K) pathway positively regulates many biological processes, including proliferation, survival, differentiation, activation, and chemotaxis, through the generation of the potent second messenger PI-3,4,5-P3 (PIP3). Consistent with its positive role in promoting proliferation and survival, activation of the PI3K pathway has been shown to be a critical event in tumor development, with many oncogenes (e.g., EGFR, Her2, and K-Ras) stimulating tumor growth by enhancing the PI3K pathway. Studies in skeletal muscle and adipocytes, where SHIP2 is highly expressed, suggest that it is an important negative regulator of insulin signaling. While SHIP1 activators in particular hold a lot of promise therapeutically, there are a number of concerns which they share with PI3K inhibitors.

All Trans Retinoic Acid, Transforming Growth Factor β and Prostaglandin E2 in Mouse Plasma Synergize with Basophil-Secreted Interleukin-4 to M2 Polarize Murine Macrophages.

In previous studies we found that macrophages (MФs) from SH2-containing inositol-5′-phosphatase (SHIP) deficient mice are M2 polarized while their wild type (WT) counterparts are M1 polarized and that this difference in MФ phenotype can be recapitulated during in vitro derivation from bone marrow if mouse plasma (MP), but not fetal calf serum, is added to standard M-CSF-containing cultures. In the current study we investigated the mechanism by which MP skews SHIP-/- but not +/+ MФs to an M2 phenotype. Our results suggest that SHIP-/- basophils constitutively secrete higher levels of IL-4 than SHIP+/+ basophils and this higher level of IL-4 is sufficient to skew both SHIP+/+ and SHIP-/- MФs to an M2 phenotype, but only when MP is present to increase the sensitivity of the MФs to this level of IL-4. MP increases the IL-4 sensitivity of both SHIP+/+ and -/- MФs not by increasing cell surface IL-4 or CD36 receptor levels, but by triggering the activation of Erk and Akt and the production of ROS, all of which play a critical role in sensitizing MФs to IL-4-induced M2 skewing. Studies to identify the factor(s) in MP responsible for promoting IL-4-induced M2 skewing suggests that all-trans retinoic acid (ATRA), TGFβ and prostaglandin E2 (PGE2) all play a role. Taken together, these results indicate that basophil-secreted IL-4 plays an essential role in M2 skewing and that ATRA, TGFβ and PGE2 within MP collaborate to dramatically promote M2 skewing by acting directly on MФs to increase their sensitivity to IL-4.

A low carbohydrate, high protein diet suppresses intratumoral androgen synthesis and slows castration-resistant prostate tumor growth in mice.

Dietary factors continue to preside as dominant influences in prostate cancer prevalence and progression-free survival following primary treatment. We investigated the influence of a low carbohydrate diet, compared to a typical Western diet, on prostate cancer (PCa) tumor growth in vivo. LNCaP xenograft tumor growth was studied in both intact and castrated mice, representing a more advanced castration resistant PCa (CRPC). No differences in LNCaP tumor progression (total tumor volume) with diet was observed for intact mice (P = 0.471) however, castrated mice on the Low Carb diet saw a statistically significant reduction in tumor growth rate compared with Western diet fed mice (P = 0.017). No correlation with serum PSA was observed. Steroid profiles, alongside serum cholesterol and cholesteryl ester levels, were significantly altered by both diet and castration. Specifically, DHT concentration with the Low Carb diet was 58% that of the CRPC-bearing mice on the Western diet. Enzymes in the steroidogenesis pathway were directly impacted and tumors isolated from intact mice on the Low Carb diet had higher AKR1C3 protein levels and lower HSD17B2 protein levels than intact mice on the Western diet (ARK1C3: P = 0.074; HSD17B2: P = 0.091, with α = 0.1). In contrast, CRPC tumors from mice on Low Carb diets had higher concentrations of both HSD17B2 (P = 0.016) and SRD5A1 (P = 0.058 with α = 0.1) enzymes. There was no correlation between tumor growth in castrated mice for Low Carb diet versus Western diet and (a) serum insulin (b) GH serum levels (c) insulin receptor (IR) or (d) IGF-1R in tumor tissue. Intact mice fed Western diet had higher serum insulin which was associated with significantly higher blood glucose and tumor tissue IR. We conclude that both diet and castration have a significant impact on the endocrinology of mice bearing LNCaP xenograft tumors. The observed effects of diet on cholesterol and steroid regulation impact tumor tissue DHT specifically and are likely to be mechanistic drivers behind the observed tumor growth suppression.